Polyunsaturated fatty acids (PUFAs) such as arachidonic acid, eicosapentaenoic acid (hereinafter referred to as “EPA” as appropriate) are contained in lipids of the cell membrane of humans, notably in the nervous system. These polyunsaturated fatty acids act as a precursor of a bioactive substance such as prostaglandin and leukotriene, and are very important pharmacological substances. In recent years, health foods containing arachidonic acid and EPA have been commercially available. In addition, fatty acids, which are used as a source material of detergents and biodegradable plastics, have captured the spotlight as material substances.
Polyunsaturated fatty acids are currently produced by extraction from cultured microorganisms or fish oil. This raises problems of high production cost, increased energy consumption and waste, and limited fish resources particularly in methods using fish oil.
The biosynthesis of arachidonic acid and EPA is believed to occur in a series of reactions involving Δ6 desaturation, chain elongation, and Δ5 desaturation, with linoleic acid and α-linolenic acid being starting materials of the reactions yielding the arachidonic acid and eicosapentaenoic acid, respectively. These reactions are catalyzed by a Δ6 fatty acid desaturase (“Δ6 desaturase” hereinafter), Δ6 fatty-acid-chain elongase (“Δ6 chain elongase” hereinafter), and Δ5 fatty acid desaturase (“Δ5 desaturase” hereinafter), respectively.
A gene for the Δ6 desaturase is cloned from several plant species. For example, the gene has been cloned from Phaeodactylum tricornutum, Physcomitrella patens, ceratodon purpureous, borage, lithospermum erythrorhizon, primrose, and anemone. Apart from plants, the Δ6 desaturase gene has also been cloned from filamentous fungi, nematodes, cyanobacteria, rats, and humans (see Non-Patent Publication 1: Eur. J. Biochem. 269, p4105, 2002; Non-Patent Publication 2: Plant J. 15, p39, 1998; Non-Patent Publication 3: Eur. J. Biochem., 267. p3801, 2000; Non-Patent Publication 4: Proc. Natl. Acad. Sci. USA 94, p4211, 1997; Non-Patent Publication 5: Lipids 37, 417, 2002; Non-Patent Publication 6: FEBS Lett. 542, p100, 2003; Non-Patent Publication 7: Whitney et al., Planta Epub 2003; Non-Patent Publication 8: Lipids 34, p649, 1999; Non-Patent Publication 9: Gene, 238, p445 1999; Non-Patent Publication 10: Biochem J. 330, p611 1998; Non-Patent Publication 11: Plant Mol. Biol., 22, p293 1993; Non-Patent Publication 12: Biochem. Biophys. res. Commun. 255, p575, 1999; and Non-Patent Publication 13: J. Biol. Chem. 274, p471, 1999). All of these Δ6 desaturates cloned from these organisms, except that obtained from cyanobacteria, have a cytochrome b5 domain at their N-terminus.
A gene of the Δ6 chain elongase was originally cloned from filamentous fungi and nematodes (see Non-Patent Publication 14: Proc. Natl. Acad. Sci. USA 97, p8284, 2000; and Non-Patent Publication 15: Proc. Natl. Acad. Sci. USA 97, p64-21, 2000). In plants, the gene has been cloned only from Physcomitrella patens (see Non-Patent Publication 16: Plant J. 31, p255, 2002).
In yeasts (Saccharomyces cerevisiae), there exist ELO2 protein and ELO3 protein, which are involved in the synthesis of a long-chain saturated acyl-chain of sphingolipids (see Non-Patent Publication 17: J. Biol. Chem., 272, p17376, 1997). The Δ6 chain elongase has an amino acid sequence homologous to the ELO2 protein and ELO3 protein. On the other hand, in plants, there exists β-ketoacyl-CoA synthase (KCS), which is another type of fatty-acid-chain elongase. This enzyme catalyzes the elongation of long-chain saturated and monounsaturated fatty acids (see Non-Patent Publication 15 and Non-Patent Publication 18: Plant Cell 7, p309, 1995). However, the KCS gene is not evolutionary related to the Δ6 chain elongase gene, or yeast ELO2 and ELO3 genes (see Non-Patent Publications 15 and 16).
A gene of the Δ5 desaturase was originally cloned from filamentous fungi (Non-Patent Publication 19: J. Biol. Chem. 273, p29360, 1998; and Non-Patent Publication 20: J. Biol. Chem. 273, p19055). The Δ5 desaturase has a cytochrome b5 domain at the N-terminus as does the Δ6 desaturase. The Δ5 desaturase gene has also been cloned from Phaeodactylum tricornutum, nematodes, rats, humans, Physcomitrella patens, and others (see Non-Patent Publication 1; Non-Patent Publication 21: FEBS Lett. 439, p215, 1998; Non-Patent Publication 22: Arch. Biochem. Biophys. 391, p8, 2001; Non-Patent Publication 23: J. Biol. Chem. 274, p37335, 1999; and Non-Patent Publication 24: J. Biol. Chem. 278, 35115, 2003).
Terrestrial plants are classified into bryophytes (Bryophyta), pteridophytes, gymnosperms, and angiosperms. Among these groups of terrestrial plants, bryophytes are known to have branched off first, and they are classified into three groups: Mosses (class Bryosida), Liverworts (class Hepaticopsida), and Hornwortz. Marchantia polymorpha is taxonomicafly closest to Physcomitrella patens among the foregoing organisms, but the latter belongs to class Bryosida while the former belongs to subclass Marchanttiidae of class Hepaticopsida. It is certain that the foregoing three groups were branched off at least about 430 million years ago. Therefore, contrary to their common name “moss,” the difference between Physcomitrella patens and Marchantia polymorpha is evolutionary far too great to be called as a difference, as compared with the difference, for example, between Arabidopsis thaliana and rice, which are believed to have branched off 200 million years ago (see Non-Patent Publication 25: www.nibb.ac.jp/˜mhasebe/Physcomitrella.html).
As a Marchantia polymorpha-derived polyunsaturated fatty acid synthetase gene, KCS-like MpFAE2 and MpFAE3 chain elongase genes have been obtained (see Non-Patent Publication 26: Biosci. Biotechnol. Biochem. 67, p605, 2003; and Non-Patent Publication 27: Biosci. Biotechnol. Biochem. 67, p1667, 2003). However, MpFAE2 and MpFAE3 are not Δ6 chain elongase genes.
As described earlier, many polyunsaturated fatty acid biosynthetic genes are cloned from various species of organisms. However, there is only a few reports in which polyunsaturated fatty acids having 20 or more carbon atoms with a degree of unsaturation 4 or greater, such as arachidonic acid and EPA, were produced in plants. As an example, it has been reported that Phaeodactyllun tricornutum-derived Δ6 desaturase gene and Δ5 desaturase gene, and a Physcomitreila patens-derived Δ6 chain elongase gene were expressed in Linum usitatissimum to produce arachidonic acid and EPA. However, this is not described in detail (see Non-Patent Publication 24).
As described earlier, polyunsaturated fatty acids, such as arachidonic acid and EPA, are produced by extraction from cultured microorganisms or fish oil. This raises problems of high production cost, increased energy consumption and waste, and limited fish resources. Polyunsaturated fatty acids such as arachidonic acid and EPA have a plurality of double bonds in the molecule. This unique characteristic enables these fatty acids to be used in various industrial products (e.g. films, biodegradable plastics, functional fabrics, lubricating oil, and material substance for detergents). By producing such polyunsaturated fatty acids in transgenic plants, it will be possible to reduce production cost and realize a more environmentally friendly production process. Once the polyunsaturated fatty acids are mass-produced with oil plants by genetic recombinant techniques, it will be possible to advantageously use such oil plants as inexpensive source materials for many different purposes.
However, in the expression of foreign genes in plants, it is difficult to predict how well the genes will function in the plants because the gene expression involves transcription, translation, and modifications. Further, in the expression of more than one foreign gene, it is envisaged that the expressed genes will function more desirably when they come from a single species of plant, as opposed to different plant species as in the case of Non-Patent Publication 24. Further, Marchantia polymorpha, which belongs to phylum Bryophyta—the first terrestrial plants—has been receiving attention as a model of higher plants, and their genes are expected to function well in other plants. Therefore, once Marchantia polymorpha-derived polyunsaturated fatty acid synthetase genes, i.e. Δ5 desaturase gene, Δ6 desaturase gene, and Δ6 chain elongase gene are obtained, it will be possible to efficiently accumulate arachidonic acid and EPA in plants by introducing these genes into plants.
The Δ5 desaturase gene, Δ6 desaturase gene, and Δ6 chain elongase gene have been cloned from Physcomitrella patens, which also belong to phylum bryophyta as does Marchantia polymorpha. However, since Marchantia polymorpha and Physcomitrella patens are evolutionary very distant species, it is not easy to obtain Marchantia polymorpha genes using Physcomitrella patens genes with the current level of technology.